Using high density tilling microarray and/or directional RNA-seq techniques, it was recently found that alternative and varying-level (dynamic) transcriptions within operons are highly prevalent and the non-coding RNA (ncRNA) and anti-sense RNA (asRNA) transcriptions are also highly pervasive. Thus prokaryotic transcriptomes seem to be more complex than previously thought. However, little is known about the patterns and rules of the transcriptomic complexity and its biological implications, as well as its underlying molecular mechanisms. Moreover, the generality of such transcriptomic complexity remains unknown because inconsistent and even contradictory results have been reported even in the same strains. Furthermore, the investigation of the transcriptomic complexity in prokaryotes has been hindered by the highly biased sequence reads of the current directional RNA-seq techniques that is further confounded in prokaryotes for the highly labile nature and extremely low concentrations of their RNAs in the cells, and the lack of an effective method for their enrichment, leading to highly non-uniform read coverage, and even numerous uncovered gaps in transcribed regions. Such highly non-uniform read coverage and prevalent uncovered gap make it very difficult to assembly full-length transcripts, let alone to detect dynamic transcriptions along operons. Consequently, little is known about the transcriptomic complexity in many medically important prokaryotes, and even in the most widely-studied model bacterium E. coli K12. This project plans to address these problems using E. coli K12 as the model system. The specific aims are: 1) to develop an algorithm and tool for sufficiently correcting the read biases in the current RNA-seq techniques; 2) to develop an accurate and efficient algorithm and tool for simultaneously assembling prokaryotic full-length transcripts and detecting possible dynamic transcriptions along the assembled transcripts using RNA-seq short reads; 3) to characterize the patterns and biological roles of alternative and dynamic operon utilizations as well as asRNA and ncRNA transcriptions in E. coli K12. Accomplishment of this project will not only further our understanding of the global architecture and complexity of the transcriptomes in E. coli K12, but also will provide the research community with computational tools and experimental methods to address the similar questions in other prokaryotes, thereby facilitating the community efforts to decipher gene regulatory networks in all sequenced prokaryotic genomes. A better understanding of the gene regulatory networks of medically, agriculturally and industriously important prokaryotes will enhance our ability to prevent and cure infectious diseases, and to produce foods and other important products.>